Measuring and Modeling The Response Characteristics Of The Environmental Phosphate Transducer In Escherichia Coli

The PhoR/PhoB two-component system in Escherichia coli is a biological transducer that senses the limitation of environmental inorganic orthophosphate, the bacteria\u27s preferred source of the essential nutrient phosphate, and transmits that information to the interior of the cell initiating a response that mitigates phosphate starvation. In the first part of this study, we present and apply a fluorescence microscopy technique to measure, in vivo, the dynamic response characteristics of the transducer with single-cell resolution. We report that the transience in the PhoR/PhoB TCS response is consistent with the transducer having a threshold sensitivity to the concentration of environmental phosphate, below which the transducer stochastically switches from a low to high operating point. Significantly, we find that the transducer response overshoots before settling to its final operating point. In the second part of this study, we investigate a series of minimal models, simple extensions of the birth-death process, that have response characteristics in common with the measured transducer response. We find that using the Hill equation as the functional form of the birth rate in an autoregulating birth-death process gives a bistable system with stochastic switching. This demonstrates that the model complexity necessary to reproduce the transducer\u27s qualitative behavior is less than the complexity of the biological system

Global wealth disparities drive adherence to COVID-safe pathways in head and neck cancer surgery

Peer reviewe

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Visualization of Content Release from Cell Surface-Attached Single HIV-1 Particles Carrying an Extra-Viral Fluorescent pH-Sensor

<div><p>HIV-1 fusion leading to productive entry has long been thought to occur at the plasma membrane. However, our previous single virus imaging data imply that, after Env engagement of CD4 and coreceptors at the cell surface, the virus enters into and fuses with intracellular compartments. We were unable to reliably detect viral fusion at the plasma membrane. Here, we implement a novel virus labeling strategy that biases towards detection of virus fusion that occurs in a pH-neutral environment—at the plasma membrane or, possibly, in early pH-neutral vesicles. Virus particles are co-labeled with an intra-viral content marker, which is released upon fusion, and an extra-viral pH sensor consisting of ecliptic pHluorin fused to the transmembrane domain of ICAM-1. This sensor fully quenches upon virus trafficking to a mildly acidic compartment, thus precluding subsequent detection of viral content release. As an interesting secondary observation, the incorporation of the pH-sensor revealed that HIV-1 particles occasionally shuttle between neutral and acidic compartments in target cells expressing CD4, suggesting a small fraction of viral particles is recycled to the plasma membrane and re-internalized. By imaging viruses bound to living cells, we found that HIV-1 content release in neutral-pH environment was a rare event (~0.4% particles). Surprisingly, viral content release was not significantly reduced by fusion inhibitors, implying that content release was due to spontaneous formation of viral membrane defects occurring at the cell surface. We did not measure a significant occurrence of HIV-1 fusion at neutral pH above this defect-mediated background loss of content, suggesting that the pH sensor may destabilize the membrane of the HIV-1 pseudovirus and, thus, preclude reliable detection of single virus fusion events at neutral pH.</p></div

Detection of single HIV-1 fusion based on release of the mCherry viral content marker.

<p>(A) Illustration of viral fusion detection with HXB2 Env pseudotyped particle (HXB2pp) co-labeled with releasable content marker Gag-imCherry (red) and core-associated marker YFP-Vpr (green). (B) HXB2 pseudovirus co-labeled with Gag-imCherry (red) and YFP-Vpr (green) fuses with CV1/CD4/CXCR4 cell as detected by instantaneous loss of mCherry signal. (C) Fluorescence intensity profiles (top) and the virus trajectory (bottom) obtained by tracking the fusing particle in panel B. (D) Fluorescence intensity profiles of another fusing HXB2pp particle. (E) Efficiency of HXB2 Env-mediated single particle fusion in CV1/CD4/CXCR4 cells untreated and treated with 10 μM AMD3100. Total number of particles examined for each condition listed at top of graph. (F) Kinetics of single HXB2pp fusion shown as a cumulative plot of the fraction of fused particles over time. Solid line is obtained by single exponential curve fitting.</p

Characterization of spontaneous content release from labeled HXB2pp adhered to coverslips.

<p>(A) Images of spontaneous content release from a single HXB2pp co-labeled with Gag-imCherry/EcpH-ICAM. Pseudoviruses were attached to poly-L-lysine coated glass coverslips in the cold and imaged in LCIB at 37°C. (B) Fluorescence intensity profiles of particle shown in panel A. (C) Comparison of content release efficiency from immobilized HXB2pp co-labeled with Gag-imCherry/EcpH-ICAM and Gag-imCherry/YFP-Vpr during 60 min image acquisition. (D) Kinetics of single content release events on poly-L-lysine coated glass and on CV1/CD4/CXCR4 cells shown as cumulative of events per total particles over time (symbols). (E) Distribution of immobilized HXB2pp on poly-L-lysine coated glass with background-subtracted EcpH intensity for all co-labeled particles (black) and co-labeled particles that spontaneously lyse (red). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148944#pone.0148944.s003" target="_blank">S3 Movie</a>.</p